Breast cancer is one of the most common cancers in women, yet even with a stabilizing incidence rate and decreased mortality, more than 180,000 women will be diagnosed and more than 40,000 women will die in the U.S. from the disease this year. A meta-analysis demonstrated an almost 2-fold increased risk for women with at least one first-degree relative diagnosed with breast cancer and there is decreased survival for breast cancer among women with a first-degree relative who also had a poor prognosis. BRCA1 and BRCA2 mutations are strongly associated with familial breast cancer however these mutations are found in only a small percentage of women with breast cancer. Recent genome-wide association studies have identified several polymorphisms associated with breast cancer although risks from these polymorphisms are modest. Our group is testing the hypothesis that certain DNA modifications are associated with breast cancer risk. Such modifications may be determined by both environmental and genetic factors, and would be expected to change over a woman's lifetime. Methylation: Our laboratory has established pyrosequencing to quantify methylation at multiple CpG sites in the promoter regions of 4 genes (BRCA1, RASSF1A, RAR Beta, and GSTP1) shown to have frequent aberrant methylation in breast cancer and, to measure global methylation, in LINE-1 repetitive elements scattered across the genome. We have recently completed the laboratory phase of a case-cohort study using Sister Study samples and are now completing data analysis. In addition we have recently completed analysis of these same samples using the Illumina 27K methylation chip. Analysis of these data has recently begun. Telomeres: Our laboratory has optimized a real-time quantitative PCR technique for estimating relative telomere length. Using this technique the number of telomere priming events, and thus the telomere amplification product, is proportional to telomere length. The method has been validated against traditional terminal restriction fragment (TRF) Southern-based analysis of telomere length, requires much less DNA, and can be efficiently done in epidemiologic studies. We are one of the first groups to employ a modified method that uses a GC clamp on the single copy gene primers in order to temperature-shift the melting point of the product to 89C. This allows us to measure both telomere and the reference single copy gene product in the same tube, thus improving the precision of the estimate and decreasing the amount of DNA required for the assay. We have adapted the method from 96- to 384-well format in order to facilitate the high throughput analysis required for epidemiology studies. We have recently completed analysis of Sister Study samples using a case-cohort design and are in the process of analyzing these data. miRNA: Altered miRNA expression is a central feature of cancer and miRNA expression signatures have been shown to be associated with diagnosis, stage, prognosis, and response to treatment. Expression patterns for cancer show high tissue specificity making them potential markers for cancer screening. Breast cancer specific miRNAs have been shown to correlate with stage, vascular invasion, proliferative index, and ER/PR status. Recently, sufficient levels of miRNAs have been found in human plasma and serum to permit profiling, with sufficient power to distinguish men with metastatic prostate cancer from men without cancer and women with ovarian cancer. Our laboratory has been working out methods to optimize miRNA extraction from serum samples and to maximize signal intensities and reproducibility in hybridizing to Affymetrix miRNA arrays. Once optimized, we plan to use Sister Study samples to study miRNA expression in women who develop breast cancer vs non-cases.